Published On: Tue, Jan 29th, 2019

A neurodevelopmental TUBB2B {beta}-tubulin mutation impairs Bim1 (yeast EB1)-dependent spindle positioning [RESEARCH ARTICLE]

INTRODUCTION

Malformation in cortical development (MCD) describes a group of severe brain malformations associated with intellectual disability and refractory infantile epilepsy. MCDs include polymicrogyria, in which an excessive number of abnormally small gyri are found in the cerebral cortex. Recently, patients with polymicrogyria associated with severe mental retardation and epileptic seizures were shown to carry a single de novo heterozygous amino-acid substitution: a phenylalanine to leucine mutation at position 265 in the conserved β-tubulin TUBB2B gene (Bahi-Buisson et al., 2014; Jaglin et al., 2009).

αβ-tubulin dimers associate to compose microtubules that display dynamicity and undergo stochastic switches between growth and shrinkage phases, the hallmark phenomenon known as dynamic instability (Alushin et al., 2014; Mitchison and Kirschner, 1984; Mitchison, 2014). This remarkable feature primarily depends on the ability of tubulins to bind and hydrolyze GTP. Microtubule elongation occurs through tubulin dimer assembling at the end of the microtubule which is capped by β-tubulin subunits – dubbed ‘plus-end’ and sometimes written ‘+end’. At growing microtubule plus-ends, GTP hydrolysis is thought to be delayed with respect to tubulin polymerization, giving rise to a protective layer of GTP-tubulin dimers, the so-called ‘GTP cap’ (Carlier et al., 1984; Dimitrov et al., 2008; Pantaloni and Carlier, 1986). The GTP cap is recognized by a subclass of proteins known as ‘plus-end tracking proteins’ (+Tips) (de Forges et al., 2016; Duellberg et al., 2016; Maurer et al., 2012). +Tips play a key role in regulating microtubule dynamics, along with numerous variables including tubulin isoforms, the amount of free αβ-tubulin dimers, molecular motors and microtubule-associated proteins (Estrem et al., 2017; Lundin et al., 2010; van de Willige et al., 2016; Vemu et al., 2017).

Several microtubule-dependent processes have been implicated in the normal folding of the six-layered human cortex. Neuronal differentiation from the neural progenitor pool depends on the orientation of the division plate, which is either aligned with or perpendicular to the ventricles, as dictated by the position of the mitotic spindle (Willardsen and Link, 2011). Later, neuronal migration involves nuclear motion (Bertipaglia et al., 2017). Spindle positioning and cell migration both universally depend on (1) dynein molecules found at microtubule plus-ends and in the cell cortex that walk along and exert force on microtubules through characteristic motor activity and (2) the actin cytoskeleton and its interaction with microtubules, as mediated by linker proteins (Coles and Bradke, 2015; di Pietro et al., 2016; Howard and Garzon-Coral, 2017).

Saccharomyces cerevisiae was one of the first organisms where the mechanisms and active components involved in controlling mitotic spindle positioning were identified before recognizing a startling conservation of the spindle orientation mechanisms and key protein partners in microtubule function between humans and budding yeast (Andrieux et al., 2017; Siller and Doe, 2009). In yeast, mitotic spindle positioning and orientation is controlled by two pathways which were identified through studies of spindle positioning relying on yeast genetics (Miller and Rose, 1998). The first pathway involves actin/Kar9 in a microtubule-guidance mechanism occurring during the S phase of the cell cycle (Lee et al., 2000; Yin et al., 2000). Kar9 links microtubules to polarized cortical actin cables by interacting with myosin-V motor and Bim1, the yeast counterpart of the +Tips protein EB1. Thus, microtubules are guided and pulled along actin cables toward the bud by the myosin-V motor (Beach et al., 2000; Hwang et al., 2003; Lee et al., 2000), resulting in spindle alignment with the mother-bud polarity axis. The second pathway involves dynein motors which power spindle movement through the mother-bud junction. This movement initially involves dynein transportation to the tips of microtubules thanks to the +Tips Bik1 (yeast CLIP170) (Carvalho et al., 2004; Caudron et al., 2008), it is then offloaded and activated at the bud cell cortex (Lammers and Markus, 2015; Sheeman et al., 2003), where it then drags the nucleus into the bud cell (Moore et al., 2009; Yeh et al., 2000).

A number of questions are raised by the discovery of the correlation between the F265L heterozygous mutation in the TUBB2B tubulin gene and a severe neurodevelopmental disorder. Is the mutant tubulin stable and incorporated into microtubules? If it is incorporated into microtubules, does it induce changes to microtubule dynamics and/or alter binding of microtubule partners? In mammalian cells, due to the large number of tubulin isotypes, it is difficult to distinguish between these possibilities and to decipher the molecular defects arising from this mutation. Indeed, in humans, each cell β-tubulin content not only results from the heterozygous expression of wild-type (wt) and F265L TUBB2B alleles, but also from the expression of other β-tubulin genes among the nine alleles present in the human genome (Findeisen et al., 2014; Khodiyar et al., 2007). Lower eukaryotes have a smaller number of tubulin genes than vertebrates; for example, the budding yeast has two α-tubulin genes (TUB1, TUB3) and only one β-tubulin gene (TUB2), and this last is highly conserved (Ludueña, 1993; Schatz et al., 1986; Thomas et al., 1985). Therefore, to gain insight into the role played by the F265 residue in β-tubulin, we produced S. cerevisiae yeast strains mutated on F265 in TUB2 and assessed the consequences of expressing this mutation as the sole source of β-tubulin. In mutant cells, mitosis and spindle orientation were impaired, and microtubule dynamics was altered. Furthermore, evidence of a reduced association of Bim1 (yeast EB1) with microtubule plus-ends was found. These results indicate that F265 in β-tubulin is essential for normal microtubule dynamics, cell division and Bim1 association with microtubule tips.

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